1. Field of the Invention
The present invention relates to compositions and methods for the high yield production of eukaryotic proteins and in particular membrane proteins, by expression of recombinant vectors designed for such high yield production in bacterial cells.
2. Background Art
Certain classes of eukaryotic, prokaryotic and viral proteins, including membrane proteins, needed in large quantities for therapeutic uses as well as for biochemical and structural studies, have proven difficult to express in recombinant systems in sufficient yields. This is particularly difficult for eukaryotic proteins with multiple membrane spanning regions including, but not limited to, G-protein coupled receptors (QPCRs) and ion channels derived from eukaryotic cells (Goeddel, 1990).
Eukaryotic membrane proteins have been expressed in a number of eukaryotic systems including mammalian cells, baculovirus systems [up to 55 pmol/mg of protein (125 xcexcg/L of culture); Loisel et al., 1997] and yeast cells (up to 14 pmol/mg membrane protein; Sander et al., 1994). However, none of these approaches has proven successful for the production of large quantities of purified eukaryotic proteins.
Furthermore, although a number of reports in the literature describe expression of eukaryotic membrane proteins such as GPCRs in prokaryotic cells (e.g., E. coli), none of these systems has proven capable of producing high levels of an intact eukaryotic protein (Table I). These bacterial cell systems have produced GPCRs in amounts of approximately several hundred receptor molecules per cell, with none of the systems producing greater than 300 receptors per cell, which corresponds to approximately 5 xcexcg protein per liter of bacterial culture.
The present invention overcomes previous shortcomings associated with high yield production of eukaryotic proteins by providing compositions and methods for producing eukaryotic proteins and in particular, membrane proteins, in high yield (i.e., at least 100 xcexcg protein/L of culture), for use in biochemical and structural studies and as therapeutic agents.